NleA suppresses formation of the inflammasome.

<p>Differentiated THP-1 cells were uninfected (UI) or infected with TOE-A5 and TOE-A5/<i>nleA</i> for 1 hr and further incubated for 3 and 6 hrs. Cells at the indicated times were immunostained, followed by visualization using a confocal microscope. The foci are indicated by arrowheads. (A) Foci formed with active caspase-1 in infected cells. The cells were stained with FAM-YVAD-FMK FLICA for active caspase-1 (Green) and DAPI (Blue). (B) Foci formed with total caspase-1 in infected cells. The cells were stained with anti-caspase-1 (Red) and DAPI (Blue). (C) Foci formed with ASC in infected cells. The cells were immunostained with anti-ASC (Red) and DAPI (Blue). (D, E and F) Quantification of respective foci formation. From nine random fields, the percentages of active caspase-1-, total caspase-1-, or ASC- foci were calculated as (number of identified speck-like structures)/(number of cells in a given field of view)x100%. Student’s t-test, * <i>p</i> < 0.05. n.d, not determined. (G) NleA reduces ASC oligomer formation. After 1 hr of infection and 3 hrs of further incubation, the cell lysates from uninfected (UI) or cells infected with TOE-A5 or TOE-A5/<i>nleA</i> were enriched for the inflammasome-containing fraction. The fraction was further treated with a DSS protein-protein cross-linker for 30 min at room temperature, and the reaction was terminated by the direct addition of 2x SDS sample buffer. The samples were separated by SDS-PAGE, and an anti-ASC antibody was used to detect ASC species. CL, cytosolic lysate.